PROJECT SUMMARY Cellular senescence is a bona fide tumor suppression mechanism but also a cause of cell and tissue aging. Senescence is caused by a range of cellular stresses and characterized by an irreversible proliferation arrest and a potent pro-inflammatory phenotype, the senescence-associated secretory phenotype (SASP). Senescence-associated proliferation arrest and SASP cooperate in tumor suppression, by arresting proliferation of damaged pre-malignant cells and promoting immune clearance of the damaged cells. However, over the longer term, as a source of chronic inflammation, SASP also promotes tissue aging and disease. Consequently, there is currently much effort devoted to development of pharmacologic approaches to eliminate senescent cells to promote healthy aging. However, these so-called senolytic drugs tend to show unwanted toxicities. An alternative, perhaps less toxic approach, is to specifically inhibit the pro-aging SASP, without impairing the tumor suppressive proliferation arrest. Hence, it is important to define the mechanism of SASP activation, because its inhibition may be an approach to combat the pro-aging effects of senescent cells. Recently, we showed that senescent cells shed fragments of nuclear chromatin into the cytoplasm via a nucleus to cytoplasmic blebbing process, so-called cytoplasmic chromatin fragments (CCF). CCF are triggers for activation of SASP in senescent cells. Specifically, CCF are sensed by the anti-viral cytoplasmic DNA sensing apparatus, cGAS and STING, to activate NFkB and SASP. Most recently, we have defined an unanticipated upstream trigger of CCF and SASP, altered mitochondria function in senescent cells. Of course, mitochondria are themselves already linked to chronic inflammation and aging. We hypothesize that formation of CCF in senescent cells is initiated by a retrograde mitochondria-to-nucleus signal that instigates a series of nuclear events, involving the DNA damage response and tight temporal and spatial control of histone modifications, culminating in expulsion of chromatin fragments from the cell nucleus into the cytoplasm. We further hypothesize that this novel signalling pathway is a target for drug interventions to suppress chronic inflammation driven by SASP in vivo, thereby potentiating healthy aging and longevity. We will test these hypotheses through three Specific Aims.